Post-transcriptional regulation of cholera toxin production in Vibrio cholerae by the stringent response regulator DksA

Expression of cholera toxin (CT), the principal virulence factor of the cholera pathogen Vibrio cholerae, is positively modulated by the RNA polymerase binding unusual transcription factor DksA (DksA(Vc)) of the stringent response pathway. Here we report that even though CT (encoded by the genes ctxAB) production is downregulated in the V. cholerae Delta dksA (Delta dksA(Vc)) mutant, the expression of the ctxA gene as well as the genes encoding different virulence regulators, namely, AphA, TcpP and ToxT, were also upregulated. Since DksA(Vc) positively regulates HapR, a known negative regulator of CT production, the increased expression of different virulence genes in Delta dksA(Vc) was due most probably to downregulation of HapR. There was no secretion/transport-related defect in Delta dksA(Vc) cells because whole cell lysates of the mutant showed a negligible amount of CT accumulation similar to WT cells. To understand further, the hapR gene was deleted in Delta dksA(Vc) background, however, the double mutant failed to rescue the CT production defect suggesting strongly towards post-transcriptional/translational regulation by DksA(Vc). This hypothesis was further confirmed when the site-directed mutagenesis of each or both of the conserved aspartic acid residues at positions 68 and 71 of DksA(Vc), which are essential for transcription initiation during the stringent response, had no effect in the regulation of CT expression. Interestingly, progressive deletion analysis indicated that the C4-type Zn finger motif present in the C-terminus of DksA(Vc) is essential for optimal CT production. Since this motif plays important roles in DNA/RNA binding, the present study indicates a novel complex post-transcriptional regulation of CT expression by DksA(Vc).